anti- p300 cbp hat antibody  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Specialised RNA decay fine-tunes monogenic antigen expression in African trypanosomes

doi: 10.1101/2025.05.24.654301

Figure Lengend Snippet: a-c , radial plots representing the proximity proteomes of 3myc TurboID-VEX2 ( a ), VEX2-TurboID 3myc ( b ) and VEX1-TurboID 3myc ( c ). Log 2 FC, which increases clockwise, represents the fold change in protein abundance between the cell lines where VEX1/VEX2 were fused with TurboID and the parental line, both treated with 50 μM of biotin for 18 hours. The y axis (-log 10 p ) represents statistical significance, which increases towards the centre. VEX1, VEX2 and CAF-1a-c are known interactors, highlighted in cyan, burgundy and yellow, respectively. Datapoints highlighted in salmon represent proteins that are significantly enriched in the TurboID +biotin samples. d, cartoon summarising the main nuclear compartments in T. brucei bloodstream forms. Known components of each nuclear body (ESB, NUFIP, SLAB or Cajal-like) in grey; new components identified in this study in salmon. e, heatmap depicting the Log 2 FC for the newly identified components of the ESB, SLAB and NUFIP body in the 3myc-TurboID VEX2 and VEX2 TurboID-3myc datasets. f-h, fluorescence microscopy analysis of ESB2 12myc ( d ), 6myc ESB3 ( e ), 6myc ESAP1 ( f ) and Pol-I RPA1 10ty in bloodstream forms. Histograms on the right-hand side depict the fluorescence signal distribution across the distance highlighted by the yellow lines. A schematic of the number of amino acids, identifiable domains and sequence-motifs is provided for each protein. PIN, RNA nuclease; RRM-like, RNA binding. i, fluorescence microscopy analysis of ESB2 12myc , 6myc ESB3 and 6myc ESAP1 in procyclic forms. j, protein blotting analysis of ESB2 12myc , 6myc ESB3 and 6myc ESAP1 expression in bloodstream (B) and procyclic (P) forms. WT, wild-type. EF1α was used as a loading control. The images in f/g/h/i (right panel) and i (left / middle panel) were acquired using a Zeiss LSM980 Airyscan 2 and a Zeiss AxioObserver, respectively, and correspond to 3D projections by brightest intensity of 0.1 μm stacks. ESB, expression-site body; No, nucleolus. DNA was stained with DAPI (cyan, grey or purple); scale bars: 2 μm. k, cartoon summarising how VEX2, ESAP1, ESB3 and ESB2 are developmentally regulated between insect (cyan) and mammalian (purple) stages of T. brucei at both RNA and protein levels. In the insect-stage, VEX2 and ESAP1 can be found throughout the nucleus in a speckle-like manner. All proteins form a single protein focus that colocalises with the ESB in the mammalian-form of the parasite. d/k, created with BioRender.com

Article Snippet: The following primary antibodies were used: mouse α-myc (Millipore, clone 4A6, 1:10,000), mouse anti-Ty (BB2, Invitrogen, 1:5,000) and mouse α-EF1α (Millipore, clone CBP-KK1, 1:30,000).

Techniques: Quantitative Proteomics, Fluorescence, Microscopy, Sequencing, RNA Binding Assay, Expressing, Control, Staining

Journal: bioRxiv

Article Title: Specialised RNA decay fine-tunes monogenic antigen expression in African trypanosomes

doi: 10.1101/2025.05.24.654301

Figure Lengend Snippet: a-b , protein blotting analysis of whole cell extracts of 3myc TurboID-VEX2 and VEX2-TurboID 3myc ( a ) and VEX1-TurboID 3myc ( b ) cell lines. An anti-myc antibody was used; EF1α, loading-control. C means clone x. c-e, fluorescence microscopy analysis of 3myc TurboID-VEX2 / Biotinylation ( c ), 3myc TurboID-VEX2 / Pol-I ( d ) and Biotinylation / Pol-I in a VEX1-TurboID 3myc cell line ( e ). Biotinylated material was detected using Streptavidin-A488. Images were acquired using a Zeiss LSM980 Airyscan 2 and correspond to 3D projections by brightest intensity of 0.1 μm stacks. DNA was stained with DAPI (cyan); scale bars: 2 μm. f-g, protein blotting analysis of immunoprecipitation samples of biotinylated material using streptavidin beads from 3myc TurboID-VEX2 and VEX2-TurboID 3myc ( f ) and VEX1-TurboID 3myc ( g ) cell lines treated or untreated with biotin. Streptavidin-HRP was used for detection. h-j, GO terms ( h, i ), protein expression and sequence homology ( j ) analyses of the 49 proteins significantly enriched in the VEX-TurboID ‘plus biotin’ samples that have not been previously associated with the ESB or surrounding nuclear bodies. h , protein network analysis based on biological process. i , similar GO terms containing functionally-related proteins cluster proximally. The size and colour of the bubbles were derived from the number of proteins in the VEX interactome that cluster in each GO term and the corresponding p value, respectively.

Article Snippet: The following primary antibodies were used: mouse α-myc (Millipore, clone 4A6, 1:10,000), mouse anti-Ty (BB2, Invitrogen, 1:5,000) and mouse α-EF1α (Millipore, clone CBP-KK1, 1:30,000).

Techniques: Control, Fluorescence, Microscopy, Staining, Immunoprecipitation, Expressing, Sequencing, Derivative Assay

Journal: bioRxiv

Article Title: Specialised RNA decay fine-tunes monogenic antigen expression in African trypanosomes

doi: 10.1101/2025.05.24.654301

Figure Lengend Snippet: a-c , Cumulative growth following tetracycline induced ESB2 ( a ), ESB3 ( b ) and ESAP1 ( c ) knockdown; * p < 0.05; ** p < 0.01, ** p < 0.001 (multiple t tests). d-f, protein blotting analysis of ESB2 ( d ), ESB3 ( e ) and ESAP1 ( f ) expression following their respective knockdowns. C1, clone 1; C2, clone 2. EF1α, loading-control. g, immunofluorescence analysis of VSG expression following ESB2/ESB3/ESAP1-knockdown (48 h); >100 cells per condition were analysed. a-c/g, error bars (not visible in g ), SD; data are averages from two independent biological replicates and representative of independent experiments. h-o, RNA-Seq analysis of ESB2 (n=5), ESB3 (n=3) and ESAP1 (n=3) knockdowns at 24 hours post induction, where n values correspond to the number of biological replicates. h, volcano plot depicting upregulation of ‘active’ and ‘silent’ ESAGs following ESB2 RNAi. i, transcript abundance at the active VSG-2 locus. Purple circle, promoter; purple box, VSG-2 ; lilac boxes, ESAGs . RPM, reads per million. Bin size 0.5 kb. j, box plots depicting ESAG transcript abundance in RPKMs (reads per kilobase per million) at the active-ES in the parental line versus ESB2 RNAi. Boxes span between the 25 th and 75 th percentile, the line corresponds to the mean value; whiskers span between minimum and maximum value; all datapoints are depicted. hyp, hypothetical. k-n, scatter plots comparing average gene expression in the parental line (in RPKMs) with fold change in expression in ESB2 RNAi 24 h / Parental ( k ), ESB2 RNAi 24 h / ESB3 RNAi 24 h ( l ), ESB2 RNAi 24 h / ESAP1 RNAi 24 h ( m ) and ESB3 RNAi 24 h / ESAP1 RNAi 24 h ( n ). o, heatmap depicting the Log 2 FC within the active-ES for induced and uninduced samples of ESB2, ESB3 and ESAP1 RNAi normalised against the parental line. ESB2 RNAi non-induced samples present some leaky expression. From top to bottom, genes are ordered as they appear in BES1 (active-ES). ESAG2 and ESAG11 were omitted as their expression could not be detected in all replicates in the parental line when applying high stringency mapping.

Article Snippet: The following primary antibodies were used: mouse α-myc (Millipore, clone 4A6, 1:10,000), mouse anti-Ty (BB2, Invitrogen, 1:5,000) and mouse α-EF1α (Millipore, clone CBP-KK1, 1:30,000).

Techniques: Knockdown, Expressing, Control, Immunofluorescence, RNA Sequencing, Gene Expression

Journal: bioRxiv

Article Title: Specialised RNA decay fine-tunes monogenic antigen expression in African trypanosomes

doi: 10.1101/2025.05.24.654301

Figure Lengend Snippet: a , Fluorescence microscopy analysis of 6myc ESB3 localisation following ESB2 and ESAP1 RNAi and 6myc ESAP1 localisation following ESB2 and ESB3 RNAi. b, ESB2, ESB3 and ESAP1 mRNA levels following VEX2, ESB2, ESB3 and ESAP1 RNAi. The values were derived from RNA-Seq data (ESB3 & ESAP1 RNAi: 3 biological replicates; ESB2 RNAi: 5 biological replicates). Bars span between minimum and maximum values; the line corresponds to the mean value, all datapoints are represented. Two-tailed paired Student’s t tests were applied; ns, non-significant; **** p < 0.0001. c-e, protein blotting analysis of 6myc ESAP1 ( c ), 6myc ESB3 ( d ) and ESB2 12myc ( e ) levels following VEX2, ESB2, ESB3 or ESAP1 RNAi at 24 hours post induction. C1, clone 1; C2, clone 2. EF1α, loading-control. f-h, fluorescence microscopy analysis of Pol-I localisation following ESB2, ESB3 and ESAP1 knockdown ( f ), ESB2 12myc , 6myc ESB3 and 6myc ESAP1 localisation following VEX2 knockdown ( g ) and 6myc VEX2 localisation following ESB3 and ESAP1 knockdown ( h ). a/f-h, all analyses were performed at 24 hours post induction. The images were acquired using a Zeiss AxioObserver and correspond to 3D projections by brightest intensity of 0.1 μm stacks. DNA was stained with DAPI (grey or cyan); scale bars: 2 μm. i-k , violin plots depicting a comparative transcriptomic analysis between VEX2, ESB2, ESB3 and ESAP1 RNAi cell lines with a focus on Pol-I transcribed gene cohorts: metacyclic VSGs ( d ), procyclins ( e ) and procyclin associated genes ( PAGs) ( f ). Log 2 FC, fold change in transcript abundance between RNAi (24 hours post induction) and the parental cell line. The violins span between minimum and maximum values, centre lines correspond to the mean, all datapoints are shown. One-way ANOVA; ns, non-significant; * p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001.

Article Snippet: The following primary antibodies were used: mouse α-myc (Millipore, clone 4A6, 1:10,000), mouse anti-Ty (BB2, Invitrogen, 1:5,000) and mouse α-EF1α (Millipore, clone CBP-KK1, 1:30,000).

Techniques: Fluorescence, Microscopy, Derivative Assay, RNA Sequencing, Two Tailed Test, Control, Knockdown, Staining

Journal: bioRxiv

Article Title: Specialised RNA decay fine-tunes monogenic antigen expression in African trypanosomes

doi: 10.1101/2025.05.24.654301

Figure Lengend Snippet: a-c ,ESB2 PIN domain analysis. a , AlphaFold2 prediction of ESB2 structure (PIN domain highlighted in purple). b-c , predicted ESB2 PIN domain superimposed with the PIN domain structure of human SMG6 (PDB ID 2HWW). Root mean square deviation (RMSD) quantifies the average distance between corresponding atoms in two superimposed structures. Catalytic residues are highlighted in c . d, CRISPR/Cas9 mediated precision editing of ESB2 D240 to alanine. No homozygous clones for the desired mutation could be generated, however successful double allele editing was achieved when using a repair template containing a synonymous mutation. 15 clones were screened for both synonymous and non-synonymous mutations, respectively. The data are representative of two independent experiments. e-i, tetracycline inducible overexpression of ESB2 12myc in bloodstream forms. e-g, fluorescence microscopy analysis of Pol-I and ectopically expressed ESB2 12myc . Wild-type or catalytic mutant (D240A, D330A, D353A), with or without an La-NLS sequence, were analysed. Images were acquired using a Zeiss LSM980 Airyscan 2 and correspond to 3D projections by brightest intensity of 0.1 μm stacks. DNA was stained with DAPI (grey); scale bars: 2 μm. The violin plot ( e ) displays data from >100 G1 cells and is representative of two biological replicates and independent experiments. The stacked graph ( f ) depicts averages of two biological replicates; >100 G1 cells per condition were analysed; error bars indicate standard deviation. The statistics depicted on the graph are focused on % of cells with an ESB (cyan), ESB + nucleolar signal (purple) and nuclear signal (salmon). Data in e-g corresponds to 24 hours post induction. h-i, protein blotting analysis of wild-type ( h ) or mutant ( i ) ESB2 expression. ‘Native’ corresponds to a cell line where ESB2 was endogenously tagged. j-m, tetracycline inducible expression of ESB2 Mut-NLS and ESB2 WT upon depletion of endogenous ESB2. j , schematics summarising the cell lines that were generated. The ectopic copies were recoded to be refractory to the RNAi target sequence and were untagged. k , cumulative growth following tetracycline induction. The ESB2 RNAi cell line was used as a benchmark. Regarding the statistical analysis, the colours indicate which cell line they refer to and correspond to induced versus non-induced conditions for each timepoint. Error bars (not visible) correspond to SD. l , RT-qPCR analysis showing successful expression of ESB2 Mut-NLS and ESB2 WT ectopic copies upon endogenous ESB2 depletion. Primers annealed in the ESB2 5’UTR and beginning of its coding sequence amplifying only endogenously expressed ESB2 (left hand side) or in the recoded region amplifying only ectopically expressed ESB2 (right hand side). The expression of the gene of interest was normalised against actin (housekeeper gene) and displayed as fold change in relation to the parental cell line. Each experiment was conducted using three technical replicates per condition, which were averaged. At least two independent clones were tested (all datapoints are displayed). m, protein blotting analysis showing the depletion of endogenous ESB2, which was C-terminally tagged with 12xmyc in the presence of ectopically expressed ESB2 Mut-NLS or ESB2 WT . Statistical analysis was performed as follows: two-tailed unpaired Student’s t test ( f ); two-way ANOVA ( g ); multiple two-tailed paired Student’s t tests ( k ); one-way ANOVA ( l ). ns, non-significant; ** p < 0.01; *** p < 0.001; **** p < 0.0001. h, i, m, EF1α was used as loading-control. The violin plot ( f ) and the floating bar graphs ( l ) span between minimum and maximum values, centre lines correspond to the mean, all datapoints are shown.

Article Snippet: The following primary antibodies were used: mouse α-myc (Millipore, clone 4A6, 1:10,000), mouse anti-Ty (BB2, Invitrogen, 1:5,000) and mouse α-EF1α (Millipore, clone CBP-KK1, 1:30,000).

Techniques: CRISPR, Clone Assay, Mutagenesis, Generated, Over Expression, Fluorescence, Microscopy, Sequencing, Staining, Standard Deviation, Expressing, Quantitative RT-PCR, Two Tailed Test, Control